Hepatitis B virus (HBV) is the infectious agent that triggers hepatitis B. Chronic HBV affects about 350 million people worldwide. Once an individual is infected, HBV targets the liver eventually causing scarring of the liver (cirrhosis) and liver failure. According to the World Health Organization, HBV is 100 times more infectious than human immunodeficiency virus (HIV) and is readily transmitted through blood and bodily fluids. There is no known cure for HBV, and even with new treatments available, each year it is estimated that 5000 Americans and one million individuals worldwide die from hepatitis's major sequelae: cirrhosis and hepatocellular carcinoma.
HBV, like all other viruses, must first attach to a cell capable of supporting its replication. Viral attachment is mediated via specific envelope proteins expressed on the surface of the virus. These proteins define the virus's tropism. Moreover, the chronic disease is associated with a massively increased risk of primary liver carcinoma which results in about one million deaths each year.
Hepatitis B virus has a small, circular DNA genome with a size of about 3.2 kb. The viral genome carries four genes named the C, S, X, and P genes. The C gene codes for the viral core protein that packages the viral genome and also for a related protein named precore protein. The precore protein is the precursor of the secreted e antigen, which may be important for the establishment of persistent infection following neonatal infection (for a review, see Ou, 1997, J Gastroenterol Hepatol., 12:S178-S187). The S gene codes for three co-carboxy-terminal envelope proteins known as surface antigens or L, M and S envelope proteins (see below). The X gene codes for a transcriptional transactivator, and the P gene codes for the viral DNA polymerase, which is also a reverse transcriptase.
The expression of the HBV genes is regulated by four different promoters and two enhancer elements (Yen, 1993, Semin Virol., 4:33-42). The core promoter regulates the transcription of the precore RNA and the core RNA, the L promoter regulates the expression of the L RNA, the major S promoter regulates the transcription of the M RNA and the major S RNA, and the X promoter regulates the transcription of the X RNA. The precore RNA and the core RNA are larger than the genome length. However, only the latter is used as the mRNA for the synthesis of the viral DNA polymerase (Nassal, et al., 1990, Cell, 63:1357-1363; Ou, et al., 1990, J Virol., 64:4578-4581). The ENI enhancer partially overlaps the X promoter, and the ENII enhancer is located upstream of the core promoter (Guo, et al., 1991, J Virol., 65:6686-6692; Shaul, et al., 1985, EMBO J., 4:427-430; Wang et al., 1990, J Virol, 64:3977-3981; Yee, 1989, Science, 246:658-661; Yuh and Ting, 1990, J Virol., 64:4281-4287). Both enhancers can upregulate the activities of all four HBV gene promoters (Antonucci and Rutter, 1989, J Virol., 63:579-583; Yee, 1989, Science, 246:658-661). Only one of the HBV DNA strands is coding, and therefore the transcription of the HBV genes is unidirectional. All of the HBV RNA transcripts terminate at the same poly(A) site in the viral genome. It has been demonstrated that cis-acting elements as well as the distance between the promoter and the poly(A) site play important roles in determining whether the poly(A) site should be used or bypassed for polyadenylation of the viral RNA (Cherrington et al., 1992, J Virol., 66:7589-7596; Guo et al., 1991, Virology, 181:630-636; Russnak and Ganem, 1990, Genes Dev., 4:764-776). For example, as this site is located less than 200 bp from the core promoter, the C gene transcripts bypass this site the first time and become polyadenylated at this site only after they have circled around the genome once and encounter the site the second time. In contrast, this poly(A) site is located approximately 2 kbp away from the S gene promoters and is therefore used efficiently by the S gene transcripts for polyadenylation when the site is first encountered during transcription. The X promoter is located about 700 bp upstream of the poly(A) site, and therefore the X gene transcripts bypass this poly(A) site with an intermediate efficiency of approximately 50% (Guo et al., 1991, Virology, 181:630-636). This leads to the production of two different X gene transcripts: one with a subgenomic size of 700 nucleotides (nt) and the other with a larger-than-genome size of 3.9 kb (Guo et al., 1991, Virology, 181:630-636).
All three Hepatitis B surface proteins, S, M and L, are also known as Hepatitis B surface antigens (HBsAg). HBsAgs are the earliest indicators of acute hepatitis B, often detectable before symptomatic onset; their expression level remains high in individuals with chronic infection. Within the HBV genome, the region encoding the HBV surface proteins contains three in-frame start sites which share a common termination codon. Because of this, the various HBV surface proteins are all related to each other by a shared region known as the S-domain. The two larger proteins (L and M, respectively) have a C-terminus in common with S. PreS2 is the sequence of M that is unique relative to S. PreS1 is the sequence of L that is unique relative to M. The N terminus of HBV preS1 contains a domain that is considered essential for an interaction between the virus and as yet unidentified host receptor(s) (Barrera, et al., 2005, J. Virol., 79:9786-9798, Engelke, et al., 2006, Hepatology, 43:750-760, Gripon, et al., 2005, J. Virol., 79:1613-1622, Loffler-Mary, et al., 1997, Virology, 235:144-152). This region overlaps with one that can act as an endoplasmic reticulum retention signal (Kuroki, et al., 1989, Mol. Cell. Biol., 9:4459-4466) probably because it interacts with host molecular chaperones (Cho, et al., 2003, J. Virol., 77:2784-2788, Ryu, et al., 2000, J. Virol., 74:110-116).
In addition to acting as HBV surface antigens/proteins, L, M and S can be secreted from infected cells as subviral particles, although L alone can only be released when S is also present (Cheng, et al., 1986, J. Virol., 60:337-344, Persing, et al., 1986, Science, 234:1388-1391). S protein alone is sufficient for N-glycosylation and secretion (Bruss, et al., 1991, Proc. Natl. Acad. Sci., USA 88:1059-1063). All three proteins undergo some level of N- or O-glycosylation prior to release, consistent with their transport through the endoplasmic reticulum (ER) and Golgi apparatus (Gelich, et al., 2005, Hepatitis B, In TOPLEY AND WILSON'S MICROBIOLOGY AND MICROBIAL INFECTIONS, Volume 2, A.S.M. Press, Washington). L is myristoylated at a glycine penultimate to the N-terminal methionine (Persing, et al., 1987, J. Virol., 61:1672-1677). This modification is not essential for assembly but is required for infectivity (Bruss, et al., 1996, Virology, 218:396-399, Gripon, et al., 1995, Virology, 213:292-299). The PreS domains of L can exist in two quite different topological conformations, due to post-translational translocation across the ER membrane (Bruss, et al., 1994, EMBO J., 13:2273-2279, Ostapchuk, et al., 1994, EMBO J., 13:1048-1057, Prange, et al., 1995, EMBO J., 14:247-256). It has been shown that sequences within S act as a signal for PreS2 translocation of M (Eble, et al., 1990, J. Virol., 64:1414-1419). Furthermore, it has been assumed that sequences within S also direct translocation of upstream sequences in the L protein (Lambert, et al., 2001, J. Biol. Chem., 276:22265-22272). Consequently, for L and M it has been presumed that the preS1 and preS2 domains, respectively, do not contain any translocation signal. As a direct test of this interpretation previous studies have attempted to fuse the preS1 plus preS2 domains to a reported protein, and were unable to detect translocation into ER-derived microsomes (Ostapchuk, et al., 1994, EMBO J., 13:1048-1057).
The major antigenic epitope of hepatitis B virus is a highly conserved region spanning 23 amino acid residues and located from amino acid position 124 to 147 of the major surface antigen. This small region designated as the group specific determinant “a” is found in all subtypes and isolates of hepatitis B viral genomes. Its antigenic properties seem due to its proposed double loop structure, to which the vaccine-induced neutralizing antibody binds.
In contrast to random mutations introduced into hepatitis B viral genomes during viral replication by the proof-reading defective reverse transcriptase, mutations induced following vaccination occur mainly in the “a” epitope of the major surface antigen. These mutant viruses are of particular interest since they show reduced affinity to the neutralizing antibody and therefore are able to replicate independently. Among these vaccine-escape mutants, the mutation at amino acid residue 145 (from Glycine to Arginine) in the second loop of the major surface antigen is the most significant because it is stable, results in conformational changes of the “a” epitope and has been reported worldwide in North America, Europe, Japan and Southeast Asia. In Singapore, for example, such mutants are the most frequent variant following vaccination. Twelve infectious variants among 41 breakthroughs have been identified as having an arginine mutation at amino acid residue 145 of the major surface antigen. There is evidence of vertical transmission from one of the 12 variants and this variant has also been associated with active liver disease. Significantly, some of these variants are now found in random asymptomatic adult population.
Hepatitis B e-antigen (HBeAg) is a hepatitis B viral protein secreted by infected hepatocytes directly into the blood only when virus is present. Usually, HBeAg in serum is associated with a high viral titre and active, progressive liver disease. As such, it is used as an indicator of an individual's ability to infect others as well as a marker to monitor efficacy of treatment. Serum HBeAg loss and development of anti-HBe antibody (seroconversion) is associated with amelioration of disease and a reduction in viral load. This also involves normalization of serum aminotransferases as well as improved histology. However, some patients may be HBeAG-negative and be anti-HBe positive yet still have high levels of virus and active disease. The latter are due to the presence of a variant form of the virus that is particularly common in the Middle East and Asia. The most common of these HBeAg mutations results in the introduction of a premature stop codon in the open-reading frame of the precore region of the virus; this leads to a lack of HBeAg secretion into the blood. Patients with precore mutant anti-HBe-positive hepatitis B have lower HBV DNA levels than HBeAg-positive patients, but they still suffer from progressive liver disease.
Hepatitis B is difficult to cure. The aim of current treatment regimens is to enable to patient's immune system to control viral infection by administering therapeutic agents that decrease HBV replication. In addition, current treatment attempts to prevent further liver damage by attenuating hepatic necroinflamation, thereby preventing progression to fibrosis. Interrupting fibrogenesis prevents subsequent progression to cirrhosis and its associated complications, thus improving survival. A typical response to treatment can be classified in three phases: (1) decrease HBV replication as measured by serum HBV-DNA levels; (2) liver necroinflamation diminishes and fibrosis is stabilized, but the risk of viral reactivation persists; (3) if the antiviral effect is sufficient (<100,000 copies of HBV DNA per ml), is maintained and is accompanied by an effective immune response with clearance of infected hepatocytes, HBe seroconversion may occur and the risk of viral reactivation is low. If HBV replication is completely interrupted with stable HBe serovonversion and loss of detectable HBsAg may occur which is associated with complete loss of necroinflamation and no risk of reactivation. This is as close to a cure as can be hoped for.
Five drugs are now licensed to treat chronic hepatitis B infection: conventional interferon alfa-2b (IFN-α 2b), peginterferon alfa-2a, lamivudine, adefovir and entecavir. However, treatment may be ineffective or become so by the emergence of drug resistant virus. Even if successful, drug regimens suffer from such disadvantages as being long-term, expensive and associated with many undesirable side effects. When end-stage liver disease supervenes in chronic HBV infection, liver transplantation is the only alternative, however viral infection persists and infects the graft, significantly shortening both patient and graft survival. In addition, while there is good agreement that combination therapy offers the best hope for an effective future strategy in disease management, improving efficacy of treatment while preventing the development of resistance, all of the combination regimens tried so far have failed to demonstrate efficacy.
After binding of the viral particles to surface molecules of hepatocytes and other cells of hepatic and nonhepatic origin, the virus is transported into the cells and the viral genome is transported into the nucleus. The viral genome, during or after entering the nucleus, is converted to a completely double-stranded supercoiled DNA genome (cccDNA). All viral RNAs are transcribed from the cccDNA. A greater than genome length RNA, the pregenome, is terminally redundant. Subgenomic RNAs are also transcribed, from which the structural and nonstructural proteins are translated.
Phosphorylation and dephosphorylation of the core protein play an important part in the assembly of the nucleocapsid, in DNA synthesis, in the association of the nucleocapsid proteins with the nuclear membrane and their transport into the nucleus, and in nucleocapsid disintegration which is necessary in order to transport the genome into the nucleus. Modifications in the phosphorylation of the nucleocapsid can interfere with the infectivity of the hepadnaviruses and with the infection process. Once DNA synthesis has reached a particular state of maturation, the virus is enveloped. Some of the nucleocapsids migrate to the nuclear membrane and thus provide the necessary increase in the cccDNA copy number.
In recent years, α-interferon produced by genetic engineering has been found useful in the treatment of HBV infections. It is a cytokine with broad antiviral and immunomodulating activity. However, it is effective in only about 33% of the patients, entails considerable side effects, and cannot be administered by the oral route.
A nucleoside derivative, Lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine), also known as thiacytidine (3TC), which has been described by Liotta et al. in U.S. Pat. No. 5,539,116, has been applied with success against HBV infection and approved by the U.S. Food and Drug Administration. It is remarkable for its high efficacy both in HbeAg-positive and HbeAg-negative patients and has few side effects. However, such therapies are associated with an increasing risk of resistance to lamivudine, which can be as high as 45-55% after the second year of treatment (Liaw et al., 2000 Gastroenterology 119: 172-180).
Although rapid decline of HBV DNA and normalization of the alanine transferase activity in serum is found in such treatment, HBV apparently cannot be completely eliminated from the liver under such therapy, so that reactivation of a hepatitis B infection is possible in many cases even after completion of a one-year treatment. Attempts are being made to prevent the above course by extending the treatment to several years, in the hope that HBV could be eliminated completely (Alberti et al., 2002, J Med Virol 67: 458-462).
In recent years considerable effort has been directed at developing safe and effective gene delivery systems and techniques. Viruses, which have evolved efficient mechanisms of delivering genomic packages to a variety of cell types, are particularly attractive candidates.
Vectors derived from retroviruses such as the lentivirus are probably among the most suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
Primate lentiviruses, such as HIV and SIV, are distinguished by their use of the CD4 protein as a receptor. They have four main genes coding for virion proteins which are, in order: 5′-gag-pro-pol-env-3′. HIV has additional genes vif, vpr, vpu, tat, rev, and nef, whose products are involved in regulation of synthesis and processing of virus RNA as well as other replicative functions.
The host range of retroviruses including lentiviral vectors can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of viral particles bearing glycoproteins (GP) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the GP were derived. Pseudotyping is a process that commonly occurs during viral assembly in cells infected with two or more viruses (Zavada, J. The pseudotypic paradox. J. Gen. Virol. 63:15-24, 1982). HIV-1 has long been known to form pseudotypes by the incorporation of heterologous GPs through phenotypic mixing, allowing an extension of the host range of HIV-1 virions beyond cells that express the CD4 receptor and an appropriate co-receptor.
However, viral pseudotyping is not a straightforward process. Co-expression of a given glycoprotein with a heterologous viral core will not necessarily give rise to highly infectious viral particles. There are examples of restriction of pseudotype formation, notably between GPs and viral cores derived from different retroviral families such as GALV and RD114 lentiviral pseudotypes. In fact, functional associations between viral cores and glycoproteins are unpredictable. An added complexity has made HBV an unlikely candidate for pseudotyping: in contrast to HIV proteins which are assembled on the cell surface, HBV envelope proteins are retained within the cell.
The occurrence of a replicative vaccine-induced HBV mutant and its ability to escape detection using standard reagents is of grave concern because it has resulted in the development of acute hepatitis B in Italy and Singapore. This situation therefore requires the urgent development of specific detection systems, as well as, effective prophylactic vaccines and antiviral agents. Thus, there is a long felt need in the art for efficient and directed means of inhibiting HBV infection and therapies to treat disorders associated thereof.